Guidance for the Clinical Management of h Fmd

A Guide to Clinical Managementand Public Health Response

for Hand, Foot and Mouth Disease (HFMD)

WHO Western Pacific RegionPUBLICATION

ISBN-13 978 92 9061 525 5

A Guide to Clinical Managementand Public Health Response


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A Guide to Clinical Management and Public Health Responsefor Hand, Foot and Mouth Disease (HFMD)

WHO Library Cataloguing in Publication Data

A Guide to clinical management and public health response for hand, foot and mouth disease (HFMD)

1.Hand,footandmouthdiseaseepidemiology.2.Hand,footandmouthdiseasepreventionandcontrol.

3.Diseaseoutbreaks.4.EnterovirusA,Human.I.RegionalEmergingDiseaseInterventionCenter.

ISBN9789290615255(NLMClassification:WC500)

World Health Organization 2011

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A Guide to Clinical Management and Public Health Response


Contents

Acknowledgements.......................................................................................v

Acronyms.......................................................................................................vi

Introduction...................................................................................................1Developingtheguide.........................................................................................1

Section1:Epidemiology................................................................................31.1Overview......................................................................................................3

1.2Descriptiveepidemiology............................................................................4

1.3Seroepidemiologicalstudy...........................................................................10

Section2:Virology........................................................................................152.1Overview......................................................................................................15

2.2Virusreceptor..............................................................................................18

2.3Recombination..............................................................................................18

2.4ReservoirofEV71.......................................................................................18

Section3:LaboratoryDiagnosis...................................................................213.1Overview......................................................................................................21

3.2Laboratorysafety.........................................................................................21

3.3Clinicalsamples...........................................................................................21

3.4Laboratorydiagnosismethods.....................................................................22

Section4:PathogenesisinEV71Infection...................................................284.1Overview......................................................................................................28

4.2Virusneuro-virulencefactors......................................................................28

4.3Hostfactors..................................................................................................29

4.4Pathologicalfindings....................................................................................30

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Section5:ClinicalFeaturesandCaseManagement.....................................355.1Casedefinitions...........................................................................................35

5.2Differentialdiagnosis...................................................................................39

5.3Clinicalassessmentandmanagement........................................................39

Section6:PreventionandControlMeasures...............................................466.1Overview......................................................................................................46

6.2PreventionMeasures:RecommendationsandRationale............................47

6.3FutureConsiderations.................................................................................51

Appendix1:SummaryofepidemiologicfindingsofHFMDfrom

surveillancedatainWesternPacificRegion(since1997)............................54

Appendix2:Benefitsandlimitations

ofspecificpreventionandcontrolmeasures.................................................62

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Acknowledgements

This document was jointly developed by the World Health OrganizationRegional Office for the Western Pacific and the Regional Emerging DiseasesIntervention(REDI)Centre.Its developmentwas coordinated byDr SatokoOtsu, (WHORegionalOffice for theWestern Pacific) and Dr Zarifah Hussain Reed (REDI Centre) with support fromDrChinKeiLee(WHOChina),DrLeVanTuan(WHOVietNam),DrHarpalSingh(WHOMalaysia),andDrWeigongZhou(CentersforDiseaseControlandPrevention,UnitedStatesofAmerica).

The following individuals contributed to chapters as lead writers, advisers or peerreviewers:

Dr Jane Cardosa, Dr Jeremy Farrar, Dr Feng Zijian, Dr Wakaba Fukushima,Dr Gao Zifen, Dr Truong Huu Khanh, Ms Keiko Kumatani, Dr Raymond LinTzer Pin, Dr Tzou-Yien Lin, Dr Ching-Chuan Liu, Dr Peter Charles McMinn,Dr Lam Yen Minh, Dr Revathy Nallusamy, Dr Nguyen Thi Hien Thanh,Dr Ooi Mong How, Dr Hiroyuki Shimizu, Dr Tom Solomon, Ms Maria Takechi,DrPhanVanTu,DrWongKumThong,DrDustinChen-FuYang.

ThanksarealsoduetoDrRuthFoxwellforprovidingtechnicalandeditorialoversight,andMsRhiannonCookforprovidingeditorialadviceandcontributingtothefinalizationofthepublication.

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Acronyms

ANS AutonomicnervoussystemBSL BiosafetylevelCA CoxsackievirusACNS CentralnervoussystemCODEHOP ConsensusdegeneratehybridoligonucleotideprimerCPE CytopathiceffectCSF CerebrospinalfluidCT ComputedtomographycAMP cyclicadenosinemonophosphateECMO Extra-corporealmembraneoxygenationEV EnterovirusHA HerpanginaHEV HumanenterovirusH&E HaematoxylinandeosinstainHFMD Hand,footandmouthdiseaseHLA HumanleukocyteantigenIgM ImmunoglobulinMIFA IndirectimmunofluorescenceassayIL InterleukinIVIG IntravenousimmunoglobulinMCP MonocytechemoattractantproteinMIG MonokineinducedbyinterferongammaPDE PhosphodiesterasePSGL HumanP-selectinglycoproteinligandRD HumanrhabdomyosarcomacellsRNA RibonucleicacidRT-LAMP Reversetranscriptionloop-mediatedisothermalamplificationRT-PCR ReversetranscriptionpolymerasechainreactionSCARB HumanscavengerreceptorclassBSD StandarddeviationUTR UntranslatedregionVTM Virustransportationmedium

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Introduction

Hand,footandmouthdisease(HFMD)isacommoninfectiousdiseasecausedby a group of enteroviruses, including Coxsackievirus A16 (CA16) andEnterovirus71(EV71). InfectionwithEV71 isofparticularconcernas itcancauseseverediseaseinchildren,sometimesresultingindeath.Overthelastdecade,manyoutbreaksofHFMDhavebeenreportedincountriesoftheWestern Pacific Region, including Japan,Malaysia and Singapore, and across China.The incidence of HFMD, particularly that caused by EV71 infection, appears to beincreasingacrosstheRegion.Thishaspromptedconcernsthat,withoutintervention,thepublichealthimpactandspreadofthediseasewillcontinuetointensify.

Thispublicationhasbeendevelopedtosupportthetreatment,preventionandcontrolofHFMD.It is intendedasaresourceforcliniciansworkingwithHFMDcasesonaregularbasis,aswellaspublichealthpersonnelwhoareresponsibleforpreventingandrespondingtooutbreaksofHFMD.ItdrawsonthemostrecentscientificliteratureandcapturesthecurrentunderstandingandexperiencesofinternationalexpertsworkingonHFMD.

Developing the guide

In2008and2009,epidemiological,diagnosticandclinicalissuesrelatingtoHFMDwerereviewedanddiscussedatthreeinternationalmeetings.Thosemeetingsemphasizedthe importanceofestablishingstandardizedsurveillancesystems that are supportedby laboratorydiagnosis,developing investigationand responsestrategies forHFMDoutbreaks,and furtheringresearch intothebestclinicalmanagement forHFMD. Inparticular, it was suggested that standardized case definitions and guidelines forclinicalmanagementofseverecaseswereneededtoassistintheoverallcontrolandmanagementofEV71-associatedHFMD.

TheWorldHealthOrganization(WHO)WesternPacificRegionalOffice,incoordinationwith the Regional Emergency Diseases Intervention (REDI) Centre, subsequentlyorganizedaninformalconsultativemeetingonHFMDinMarch2010inKualaLumpur,Malaysia.Seventeenregionalandinternationalexpertsattendedthemeeting.Findingswere summarized and recommendations developed in the areas of: surveillance,epidemiology and burden of disease; characterization of etiological agents and

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transmission; pathogenesis; laboratory diagnosis; clinical features andmanagement;andpreventionandcontrol.

In July 2010, theREDICentre invited 10 clinicalmanagement experts to a furthermeetingonHFMDinSingaporetoreviewthedraftguidancedocumentandconsolidateup-to-dateknowledgeandexperiencesontheclinicalmanagementofHFMDcausedbyEV71.

TheresultingdocumentfromtheabovetwomeetingshasbeenreviewedbyexpertswithinandoutsidetheWesternPacificRegion,andconsensusreachedonthecontentofeachchapter.

The support of all thosewho contributed to development of this guide is gratefullyacknowledged.

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Section 1: Epidemiology

1.1 Overview

Many small and large outbreaks associatedwith EV71 infection have beenreportedthroughouttheworldsincetheearly1970s(1,2).Childrenhavebeenmostcommonlyaffectedinthoseoutbreaks,andclinicalmanifestationofcaseshasbeenmostlytypicalofHFMD,withfever,skineruptionsonhandsandfeet,andvesiclesinthemouth.However,casesinvolvingthecentralnervoussystem(CNS)and/orpulmonaryoedemahavealsobeenobserved(3).

In the Western Pacific Region, widespread epidemics have been reported in manycountries, includingAustralia,BruneiDarussalam,China, Japan,Malaysia,Mongolia,theRepublicofKorea,Singapore,andVietNam.Severalcountrieshavealsoreportedfatalcases,withsevereCNSdiseaseorpulmonaryoedema.In2009,forexample,anoutbreak inmainlandChina involved 1 155 525 cases, 13 810 severe cases and 353deaths.

LessisknownregardingthedescriptiveepidemiologyofHFMDorEV71infectionincountriesoutsidetheWesternPacificRegion.AlthoughdozensoffatalitieswithCNSinvolvementwerereportedduringEV71outbreaksinBulgariain1975andHungaryin1978,therehavebeenfewfatalcasesreportedoverthelastthreedecades.Arecentlongitudinal study fromNorway suggested asymptomatic circulation of EV71 in thecommunity.

Information regarding the recent seroepidemiology of EV71 in the Western PacificRegion is limited.A cross-sectional study in Singapore indicated that, following thedeclineofmaternalantibodies,theseroprevalenceforEV71increasedatanaveragerateof12%peryearinchildrenfromtwotofiveyearsofage,andreachedasteadystateofapproximately50%inthoseagedfiveyearsorolder.Similarresultsusingthreegroupsofstoredserawerefoundinacross-sectionalstudyperformedinTaiwan(China).TwootherseroepidemiologicalstudiesinTaiwan(China)indicatedthat,followingadecreasein the circulation of EV71 in the community, an accumulation of susceptible youngchildrenmayhavecontributedtothelarge-scaleepidemicthatoccurredtherein1998.

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1.2 Descriptive epidemiology

DiseaseassociatedwithEV71infectionwasfirstdescribedbySchmidtandcolleaguesin1974,whoreportedon20patientswithCNSdisease,includingonefatalityinCalifornia,UnitedStatesofAmerica,between1969and1972(1).SubsequentoutbreaksassociatedwithEV71infectionwerereportedinNewYork,UnitedStatesofAmerica,in1972and1977(4-6),Australiain1972-1973and1986(7,8),Swedenin1973(9),Japanin1973and1978(10-13),Bulgariain1975(14,15),Hungaryin1978(16,17),Francein1979(18),HongKong(China) in1985(19),andPhiladelphia,UnitedStatesofAmerica in1987(20). During those outbreaks,EV71 caused awide spectrumof diseases, includingHFMD, aseptic meningitis, encephalitis, paralysis, acute respiratory symptoms andmyocarditis.

Thoseoutbreaksreportedbetween1974andthemid-1990smaybeclassifiedaseitherbenignorsevereinnature(21).Fortheformer,typicalexamplesincludethelargeoutbreaks inJapan in1973and1978, involving3296and36301cases,respectively.Although cases involving CNS were observed during those outbreaks, including anumberof fatalities,clinicalmanifestationofcaseswasmostlytypicalofHFMD(10-13).ThatwasalsothecasefortheoutbreakinAustraliain1986,althoughnofatalitieswere reported (8). Reports of other outbreaks, however, have contained significantcomponentsofCNSdisease.AlargeepidemicinBulgariain1975,involving705casesandincludingalargenumberoffatalities,wasinitiallythoughttorepresentpoliomyelitisorencephalitisandwasnotcharacterizedasHFMD(14).AsimilarepidemicofacuteCNSdiseaseinHungaryin1978showedthatonlyfourcaseswereclassifiedasHFMDamong323caseswithEV71infection(17)

Inthelate1990s,twowidespreadcommunityoutbreaksassociatedwithEV71infectionoccurred;thefirstinSarawak,Malaysia,in1997,andthesecondinTaiwan(China)in1998,with2628and129106casesreported,respectively(22,23). Althoughclinicalmanifestations during those outbreaks were mostly typical of HFMD, a cluster ofdeathsamongyoungchildrenwasidentified.Caseswithrapidlyprogressiveandfatalpulmonaryoedema/haemorrhagewerealsoobservedforthefirsttime

Numerous Member States in the Western Pacific Region have since experiencedlargeHFMDepidemics associatedwithEV71 infection. Several countries have alsoreportedsubstantialnumbersofdeaths.Thefollowingsectionpresentsthedescriptiveepidemiologyofanumberofthoseepidemics.Inaddition,findingsfromthesurveillancedataofselectedcountriesintheWesternPacificRegionsince1997aresummarizedinAnnex1.

Australia

Between February and September 1999, 14 cases of EV71-associated neurologicaldiseasewere identifiedatahospitalduringacommunity-wideoutbreakofHFMDin

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Perth,WesternAustralia.Twelve(86%)ofthe14childrenwerelessthanfouryearsofage(24).

AnoutbreakofHFMDduetoEV71occurredinSydneyinthesummerof20002001.Approximately 200 children presented to hospital, including nine patientswithCNSdisease and five with pulmonary oedema. EV71 was identified in all patients withpulmonaryoedema(25).

Brunei Darussalam

Brunei Darussalam experienced its firstmajor reported outbreak of EV71 betweenFebruaryandAugust2006. More than1681childrenwere reportedlyaffected,withthreedeathsresultingfromsevereneurologicaldisease.EV71wasisolatedinsamplesfrom34ofatleast100patientsdiagnosedwithHFMDorherpangina(HA),includingtwopatientswhodiedasaresultofsevereneurologicalcomplications(26).

China

Between March and May 2007, an outbreak of HFMD occurred in Linyi City,ShandongProvince,China.By22May2007,1149caseshadbeenreportedthroughacountrywide disease-reporting system in mainland China. The majority of thosepatients(84.4%)wereyoungerthanfiveyearsofage.Eleven(0.9%)oftheHFMDcaseswereclassifiedassevere,presentingwithneurologicalcomplications. Three (0.3%)children(agedthreeyearsoryounger)diedduringtheoutbreak.Atotalof233clinicalspecimenswere collected from105 hospitalized patients, including 11 patientswithsevereHFMD.Amongthose,55(52.4%),includingsixseverecases,wereconfirmedtobeEV71infections(27).

Between1Januaryand9May2008,61459HFMDcasesand36deathswerereportedthrough Chinas disease reporting system. However, prior to 2May 2008, HFMDwasnotcategorizedasanotifiablediseaseandreportingofHFMDreliedonvoluntaryreportssubmittedbyclinicians.ThenumberofreportedcasesincreasedsharplyafterthediseasewasdesignatedasaclassCnotifiabledisease,withcasesbeingreportedfromnearly all provinces.The five provinceswith the highest numbers of reportedcaseswereGuangdong(11374),Anhui(9235),Zhejiang(6134),Shandong(4566)andHenan(3230).Childrenyoungerthanfiveyearsofageaccountedfor92%ofreportedHFMDcases.Among582samplestested,EV71accountedfor54.5%ofcases(28).

More detailed studies of the 2008 outbreakwere reported fromFuyangCity,AnhuiProvince,where6049caseswere reportedbetween1March and9May 2008. Ofthose,353(5.8%)weresevereand22werefatal(casefatalityrate:0.4%).Amongthereportedcases,themale-to-femaleratiowas1.9:1andtheagerangedfrom28daysto18years,with78%ofthecasesbeingthreeyearsofageoryounger.Epidemiologicalinvestigation revealed no contact between the 22 fatal cases, but environmentalinvestigationofthehouseholdsofthefatalcasesrevealedpoorhygieneandsanitary

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conditions. The initiallyhighcase-fatalityrate(2.9%(18/610)between1Marchand23April2008)wasattributedto:rapiddiseaseprogression;lateclinicalpresentation;and limited localmedical capacity. The case-fatality rate decreased considerably (to0.07% (4/5439) for the period between 24April and 9May 2008) once the etiologyofthediseasewasknownandearlytreatmentwasprovidedtoseverepatients.Thatwasattributedtoenhancedsurveillanceandimplementationofpreventionandcontrolmeasures(28).Itshouldbenotedthat,duringtheoutbreak(asof2May2008),HFMDwasdesignatedasaclassCnotifiabledisease.

In2009,thenumberofHFMDcasesnotifiedinmainlandChinaamountedto1155525,including13810(1.2%)severecasesand353(0.03%)deaths.Themale-to-femaleratiowas1.8:1.Ofthosecases,93%werefiveyearsofageoryounger,and75%werethreeyearsofageoryounger.ThecaseswerewidelydistributedacrossChinaandincludedbothclinicallydiagnosedandlaboratory-confirmedcases.Forthelaboratory-confirmedcases,EV71wasresponsiblefor41%ofthecases,81%oftheseverecasesand93%ofthedeaths.

Taiwan (China)

In Taiwan (China), HFMD/HA has been included in the national sentinel-physicianreporting systemsince3March1998due to theprevalenceofHFMD/HAcases. In1998,129106HFMD/HAcaseswerereported in twowavesbetween29Marchandtheendoftheyear. Theyincluded405(0.3%)patientswithseveredisease,mostofwhomwerefiveyearsofageoryounger.Seventy-eight(19.6%)patientswithseverediseasedied,71(91%)ofthemfiveyearsofageoryounger.Ofthepatientswhodied,65(83%)hadpulmonaryoedemaorpulmonaryhaemorrhage.EV71wasfoundin44of59(75%)isolatesfrompatientswithsevereinfectionswhosurvived,while34of37(92%)isolatesfrompatientswhodiedwerepositiveforEV71(23).Furthersmalleroutbreaksoccurredin2000and2001,involving291and389severecasesand41and55fatalities,respectively (41). Sentinel physician surveillance data for 2000 and 2001 indicatedsimilar levels of diseasewere caused byCA16 andEV71 (17.1% and 18.2% versus15.5%and15%,respectively). EV71wasassociatedwith47.3%(26/545)ofcasesin2000andwasthedominantstrainassociatedwithfatalitiesin2001(25/41,61%).

Between1998and2005,thenumberofsevereHFMD/HAcasesperyearrangedfrom35to405.Ofthe1548severecasesidentifiedduringtheeight-yearperiod,93%werefouryearsofageoryounger,and75%weretwoyearsofageoryounger.Themale-to-femaleratiowas1.5:1.Atotalof245fatalcaseswerereportedduringthesameperiod.EV71positivityratesamongthefatalcasesrangedfrom11%to100%ineachyear(42-44).Thenumberofseverecasesanddeaths,respectively,were:11and0in2006;12and2in2007;373and14in2008;and29and2in2009.

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Japan

Approximately2400paediatricclinicsparticipatedinthesentinelsurveillancesystemin Japan between 1993 and 1998,with an average of 36.5 cases ofHFMD reportedper sentinel site annually (29). Since 1999, approximately 3000 paediatric clinicshaveparticipatedinthesentinelsurveillancenetwork,withanaverageof42.7casesofHFMD reported per sentinel site between1999 and 2005.Large-scale outbreaksoccurredin2000and2003,withthenumbersofreportedcases(casespersentinelsite)205365(68.96)and172659(56.78),respectively.Approximately90%oftheHFMDcaseswereagedfiveyearsofageoryounger(30).

HFMD is also included in the InfectiousAgentsSurveillance system in Japan. TheInfectious Agents Surveillance reports are derived from approximately 10% of thesentinelclinicsandallthesentinelhospitalsinthenetwork.EV71wasfoundtobetheprimary causative agentofHFMDepidemics inboth2000and2003 (30). AlthoughthereisnosurveillancesystemforsevereorfatalcasesofHFMDinJapan,suchcaseshavebeenidentifiedthroughcase-seriesfromhospitalsduringepidemicperiodsinthecommunity. In1997, threedeathsofyoungchildren fromHFMDorEV71 infectionwereidentifiedinOsakaprefecture(30).Inthesummerof1997,12patients,agedtwoweeks to six years,with serologically confirmedEV71 infections,were hospitalizedin Otsu city as a result of CNS involvement (31). Between June and August 2000,30 caseswithHFMD complicated byCNS involvementwere hospitalized inHyogoprefecture.Onepatientagedtwoyearsdiedasaresultofpulmonaryoedemacausedbybrainstemencephalitis.EV71was isolated fromnine (69%)of 13 faecal samples,includingasamplefromthefatalcase(32).Anationwidequestionnairesurveyfound272complicatedcaseswithHFMDduringtheperiod2000-2002.Ofthese,226casesoccurredin2000,32in2001,and14in2002.Therewerefourcasesinvolvingsequelaeandonefatalcasereportedin2000(52).

Malaysia

InSarawak,Malaysia,awidespreadcommunityoutbreakofHFMD,primarilycausedbyEV71infection,beganinearlyApril1997.From1Juneto30August1997,atotalof2628caseswerereportedtotheSarawakStateDepartmentofHealth.Duringtheoutbreak,889childrenwerehospitalized,including39patientswithasepticmeningitisoracuteflaccidparalysis. Atotalof29previouslyhealthychildrenyoungerthansixyearsofage(median,1.5years;range,0.55.9years;male-to-femaleratioof1.9:1)diedofrapidlyprogressivecardiorespiratoryfailure.EV71wasisolatedinsamplesfromsixofthefatalcases(22).Laterin1997,anoutbreakinvolving4625hospitaladmissionsoccurredinpeninsularMalaysia,resultingin11fatalcases(35).

AsentinelsurveillanceprogrammeforHFMDwassubsequentlyestablishedinSarawakinMarch1998.BetweenMarch1998andJune2005,4290specimenswerecollectedfrom 2950 children, with a male-to-female ratio of 1.4:1. During that period, two

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largeoutbreakswereidentifiedinSarawak,in2000and2003.EV71wasthedominantenterovirus serotype of the isolates in both years (36). The sentinel surveillanceprogrammeinSarawakisongoing,withtwomoreoutbreaksidentified:thefirstin2006andthesecondin2008/2009.

Mongolia

OfficialreportingofHFMDinMongoliabeganin2008,with3210casesreportedduringthatyear.Caseswereequallydistributedbetweenthecapitalcityandtheprovinces.Amongthe245samplesinvestigated,102(41.6%)werepositiveforEV71(37).

Republic of Korea

TheincidenceofHFMD/HAincreasedintheRepublicofKoreaduring2000,withanoutbreakofHFMD/HAinChejuProvince.WhiletheactualnumberofcasesofHFMD/HAisunknown,nofatalitywasassociatedwiththeoutbreak(33).

AsentinelsurveillancesystemforEVinfectionwasinitiatedin2005.BetweenJanuary2008and30October2009,719suspectedcasesofHFMDorHA(200casesin2008and519casesin2009)wereidentified,includingonefatalcaseresultingfromsevereneurological complications and two cases resulting in a comatose state.Enteroviruswasdetectedin447(62.2%)cases.Ofthose,enteroviralgenotypewasidentifiedin218cases(53casesin2008and165casesin2009).In2008,themostcommonpathogendetectedwasCA10 (18 cases; 34.0%),while, in 2009,EV71was themost commonpathogendetected(91cases;55.2%)(34).

Singapore

AlargeepidemicofHFMDcausedbyEV71infectionoccurredinSingaporebetweenSeptemberandOctober2000,with3790casesreportedandthreedeaths.Ofthe104patientswhowereclinicallydiagnosedwithHFMDandwhosesamplesyieldedatleastonevirus,EV71wasthemostcommonlyisolatedvirus(73%)(38).

ReportingHFMDsubsequentlybecamemandatoryinOctober2000.Intheseven-yearperiodfrom2001through2007,nationwideepidemicsofHFMDwereobservedin2002(16228reportedcases),2005(15256reportedcases),2006(15282reportedcases)and2007(20003reportedcases).Theage-specificannualincidenceratewashighestin those aged 04 years, ranging from1640.5 to 5975.5 per 100 000 population andaccountingfor62.2%to74.5%ofreportedcases.DuringtheEV71-associatedHFMDepidemicinMarchandApril2006,1.8%ofthecaseswerehospitalized.Thatratewasmorethantwiceashighas inthoseepidemicscausedbyCA16(betweenMarchandApril2005,0.8%ofcaseswerehospitalized,andbetweenAprilandMay2007,0.7%ofcaseswerehospitalized).

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InasmalleroutbreakinJanuaryandFebruary2001,5187caseswerereported,includingthreeHFMD-associateddeaths.Ofthefatalcases,onewasagedfouryears,whiletheothertwowereboth11monthsofage(39).

BetweenlateMarchandMay2008,anationwideepidemicofEV71-associatedHFMDoccurred.Inthefirst24weeksof2008,thenumberofreportedHFMDcaseswas15030,atwo-foldincreasecomparedwiththesameperiodin2006(themostrecentEV71-associatedepidemicpriorto2008).Fortheepidemicperiodfromweek8toweek24in2008,EV71constituted33.2%ofthesamplestested.EV71positivitywassignificantlyhigherduringthe2008epidemicperiodthanin2006(40).

Viet Nam

InsouthernVietNam,anoutbreakofacuteencephalitisassociatedwithHFMDwasreportedinHoChiMinhCityin2003.In2005,764childrenwerediagnosedwithHFMDinHoChiMinhCitythroughsentinelsurveillanceatthelargestpaediatrichospital,withmostcases(96.2%)beingfiveyearsofageoryounger.AllpatientsprovidedspecimensandHEVwas isolated from 411 patients. Of those, 173 (42.1%)were identified asEV71,and214(52.1%)asCA16.OfthosepatientswithEV71infections,51(29.3%)werecomplicatedbyacuteneurologicaldiseaseandthree(1.7%)werefatal(45).

In20062007,sentinelsurveillanceatthesamehospitalreported305casesdiagnosedas neurological disease, of which 36 cases (11%), and three deaths (0.01%), wereassociatedwithEV71.

In2007,2008and2009, thenumbersofreportedand fatalcaseswere:5719and23,10958and25,and10632and23,respectively.Themajorityofthecaseswereinthesouthernpartofthecountry.

In northern Viet Nam, EV71/C4 has only been identified in one patient with acuteencephalitis since 2003. Between 2005 and 2007, EV71/C5 was identified in sevenpatientswithacuteflaccidparalysis.Allcaseswereunderfiveyearsofage.During2008,88casesofHFMDwerereportedfrom13provinces.Theresultsofvirusisolationfromthe88casesconfirmedthat33 (37.5%) isolateswereenterovirus-positive, includingnine(27.3%)withEV71,23(69.7%)withCA16,andonewithCA10.Nosevereorfatalcaseswerereported.Themajorityofcaseswereunderfiveyearsofage.

Countries outside the Western Pacific Region

Less is known regarding the descriptive epidemiology of HFMD or EV71 infectionin countries outside theWestern Pacific Region. In TheNetherlands, only severe,hospitalizedcasesofEV71infectionarereportedaspartof thenationalsurveillancesystem.Whilebetween1963and2008therewasnoindicationofEV71-relatedfatalities,58casesofEV71infectionrequiringhospitalizationwerereportedin2007aftera21-yearperiodoflowendemicity(46).

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In the United Kingdom, there is evidence of continuous circulation of EV71, withEV71isolatedeachyearfrom1998to2006,exceptfor2003.Of32patientswithEV71infectionaccompaniedbyneurologicalcomplicationsand/orcutaneousmanifestationsduringthateight-yearperiod,onehadfatalencephalitis(47).

AlongitudinalstudyfromNorway,conductedbetweenSeptember2001andNovember2003,indicatedasymptomaticcirculationofEV71.Atotalof113healthyinfantsfromtheageofthreemonthswererecruitedtoprovidemonthlystoolsamplesandclinicaldataupuntiltheageof28months.PrevalenceofEV71instoolsamplesshowedthatEV71wascirculatingwidelybetweenOctober2002andOctober2003.However,datafromasurveillance/registersystemshowednocorrespondingincreaseinthenumberofhospitalizedpatientswithencephalitis,HFMDorHAwithintheagegroupduringthesameperiod(48).

1.3 Seroepidemiological study

Recent data regarding the seroepidemiology of EV71 in theWestern Pacific Regionarelimited.InSingapore,aserologicalsurveywasconductedatapaediatricclinicattheNationalUniversityHospitalthatincludedallchildrenbornatthehospitalorthoseaged12yearsoryoungerbroughtforroutinevisitsandvaccinationsbetweenJuly1996andDecember1997,givingatotalof856children.Antibodyprevalenceincordbloodsuggestedthat44%ofmothershadantibodiestoEV71.NoneofthechildrentestedhadmaternalantibodiestoEV71afteronemonth,andantibodieswerefoundinonlyoneofthe124samplesfromchildrenaged1-23months.Inchildrenagedfromtwotofiveyears,theseropositiverateincreasedatanaverageof12%peryear.Insamplesfromchildrenfiveyearsofageandolder,theage-specificseroprevalencesteadiedatapproximately50%(49).

Anothercross-sectionalstudycarriedoutinTaiwan(China)examinedseroprevalenceratesforEV71byusingamicro-neutralizingassaytotestthreegroupsofstoredseracollectedin1994,1997and1999.Regardlessoftheperiodfromwhichthespecimensweretaken,theseropositiverateswererelativelyhigh(38%-44%)ininfantsyoungerthan sixmonthsof age. The rates declined to 0%-15% in infants age7-11months,increasedgraduallythereafteruntiltheageofsix,andreachedaplateauatabout50%inchildrenolderthansixyearsofage(50).

Two seroepidemiological studies have contributed to the understanding of factorsunderlyingtheHFMDoutbreakinTaiwan(China)in1998.Inacross-sectionalstudy,neutralizing antibodies toEV71were assayed for 539 subjectswho provided serumsamples for vaccine trials or health examinations in twohospitals between July andDecember 1997. Age-specific EV71-seropositive rates before the outbreak wereinverselyrelatedtoage-specificmortalityratesandsevere-caseratesinthecommunityduringtheoutbreak(r=-0.82and-0.93,respectively)(51).Alongitudinalstudywasalsoconductedusingserumsamplesfromabirthcohortstudyof81healthychildren

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whoprovidedyearlybloodsamplesbetween1988and1998.Thestudyfoundthattheyearly incidence ofEV71 seroconversionwas 3%-11%between 1989 and 1997, andthat68%of childrenhad serological evidenceofEV71 infectionby1997. However,comparedwith previous years, the seroconversion rate forEV71was relatively lowbetween1994and1997.TherarityofEV71infectionbetween1994and1997suggestsdecreasedcirculationofEV71inthecommunity.Anaccumulationofsusceptibleyoungchildrenmaythereforehavecontributedtothe large-scaleepidemicthatoccurredin1998(44,50).

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10. Tagaya I, Tachibana K. Epidemic of hand, foot and mouth disease in Japan, 19721973:

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11. HagiwaraA,TagayaI,YoneyamaT.Epidemicofhand, footandmouthdiseaseassociated

withenterovirus71infection.Intervirology,1978,9(1):6063.

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incidence of complication disorders of central nervous system. Archives of Disease in

Childhood,1980,Aug,55(8):583588.

13. TagayaI,TakayamaR,HagiwaraA.Alarge-scaleepidemicofhand,footandmouthdisease

associated with enterovirus 71 infection in Japan in 1978. Japanese Journal of Medical

ScienceandBiology,1981,Jun,34(3):191196.

14. Shindarov LM, et al. Epidemiological, clinical, and pathomorphological characteristics

of epidemic poliomyelitis-like disease caused by enterovirus 71. Journal of Hygiene,

Epidemiology,MicrobiologyandImmunology,1979,23(3):284295.

15. Chumakov M, et al. Enterovirus 71 isolated from cases of epidemic poliomyelitis-like

diseaseinBulgaria.ArchivesofVirology,1979,60(3-4):329340.

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17. NagyG,etal.Virologicaldiagnosisofenterovirustype71infections:experiencesgained

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PediatricInfectiousDiseaseJournal,1987,Feb,6(2):206-208.

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outbreakofhand,foot,andmouthdiseaseinWesternAustralia.ClinicalInfectiousDiseases,

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31. KomatsuH,etal.OutbreakofsevereneurologicinvolvementassociatedwithEnterovirus

71infection.PediatricNeurology,1999,Jan,20(1):1723.

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mouthdiseaseinJapanduringthesummerof2000:detectionandmolecularepidemiologyof

enterovirus71.MicrobiologyandImmunology,2002,46(9):621627.

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southernVietnam,2005.EmergingInfectiousDiseases,2007,Nov,13(11):17331741.

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from1998to2006.JournalofClinicalMicrobiology,2008,Oct,46(10):31923200.

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109(6):e88.

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2010Apr;52(2):2037

[ 15 ]



Section 2: Virology

2.1 Overview

ThemajoretiologicalagentsthatcauseHFMDarethehumanenterovirusesspeciesA(HEV-A),particularlycoxsackievirusA16(CA16)andenterovirus71(EV71). Thesebelong to the genus Enterovirus within the family Picornaviridae. Other HEV-Aserotypes,suchasCoxsackievirusA6andCoxsackievirusA10,arealsoassociatedwithHFMDandherpangina. While all these viruses can causemild disease in children,EV71hasbeenassociatedwithneurologicaldiseaseandmortalityinlargeoutbreaksintheAsiaPacificregionoverthelastdecade(14).

Enteroviruses are small viruseswith virions that are about 30 nm in diameter andcomposedoffourstructuralproteinscalledVP1,VP2,VP3andVP4.VP1isthemajorcapsidproteinonthesurfaceofthevirion,whileVP4isnotexposedonthesurface.Serotypingofhumanenteroviruseshastraditionallybeenbasedonneutralizationtestsusing specific antiserum pools: as such they are directed particularly at serologicalresponsestotheVP1protein.Morerecently,dueinparttolimitedaccesstoserotypingantisera and improved accessibility tomolecular technology, there has been amovetowardsusingmoleculartypingmethods.ThegeneencodingVP1isthetargetgenemostoftenusedinmoleculartypingmethodsforenteroviruses. It isforthisreasonthat a wealth of genetic sequence data on EV71 is available, enabling the geneticclassificationofvirusstrainsincommoncirculation(57).

Ribonucleic acid (RNA) viruses, such as the enteroviruses, generate evolutionarychangesfairlyrapidly.Labels(genogroups)canbeappliedtoclustersofepidemiologicallyrelatedEV71strains,generallythosewithuptoa5%nucleotidedifferenceintheVP1region. These genogroup divisions have no known significance for any enterovirusotherthanbeingaconvenientlabeltoreflectviralsequenceclusteringwithinaseaofgeneticdiversity.ItisimportanttonotethatthereisnotyetevidenceofanyassociationbetweenvirulenceandparticulargenogroupsorsubgenogroupsofEV71.

Figure 1 on the succeeding page shows a phylogenetic tree containing the threegenogroupsofEV71:genogroupsA,BandC.TheprototypeEV71virusisBrCr,isolatedinCaliforniain1969.ThisistheonlysampledvirusofgenogroupA(8).

[ 16 ]


Figure 1: Circulating genotypes of HEV71 between 1970 and 2010

HEV71 - circulating genotypes, 1970 - present

White Dendrograms:Worldwide, 1970-1995*C1 has continued to circulate in the Asia-Pacific region to the presentColoured Dendrograms:Asia-Pacific, 1997-present

A (BrCr)

C1* Singapore 2000, Sarawak WA 2001 & 2005, Vietnam 2005

C2 Taiwan 1998 WA 1999

C3 Korea 2000

C4 China & Korea, 2004, Vietnam 2005

C5 Vietnam, 2005-06

B1

B2

B3 1997-99

B4 2000-01

B5 2003,2006

B6 2006

Southeast Asia

1997-2006

SubgenogroupB1wascirculatingintheUnitedStatesofAmerica,Europe,JapanandAustraliainthe1970s,whilesubgenogroupB2wassampledmainlyintheUnitedStatesinthe1980s.In1997,whenthefirstofseveralrecentandlargeAsiaPacificoutbreaksofEV71occurredinSarawak,Malaysia,themajorcirculatingviruswasaBgenogroupvirus thatwas clearlydistinct from thegenogroupBviruses thathadbeensampledinthe1970sand1980s.ThiswasthennamedsubgenogroupB3.TheviruswasalsofoundintheMalaysianpeninsulaandinSingapore(1,2).

In1998, another largeoutbreakofEV71HFMDoccurred in theAsiaPacific region,thistimeinTaiwan(China).ThemajorcirculatingviruswasfromsubgenogroupC2.ThissubgenogrouphadalsobeenfoundinJapanandwasassociatedwithanoutbreakinPerth,WesternAustralia,in1999,wheregenogroupB3viruseswerealsocirculating.InTaiwan(China)in1998,subgenogroupB4viruseswerealsosampledand,by2000,thoseviruseshadbeenassociatedwithlargeoutbreaksthroughouttheregion,includinginJapan,MalaysiaandSingapore,aswellasinTaiwan(China),wheresubgenogroupB4virusesreplacedtheC2viruses.InSarawak,Malaysia,subgenogroupB4wasreplacedbysubgenogroupB5in2003,andthishassinceremainedthedominantsubgenogroup.SubgenogroupB5virusesarealsoimportantcirculatingvirusesinJapan,SingaporeandTaiwan(China)(13,921).

[ 17 ]



SubgenogroupC1 viruseswere sampled inNorth America in the 1970s and 1980s.Throughoutthe1990s,anduptothepresentday,theyhavebeenconsistentlyidentifiedinmanypartsoftheworld,includinginAustralia,Japan,Malaysia,NewZealand,Norway,ThailandandtheUnitedKingdom,Despitebeingfoundincirculationinmanycountries,however,thesubgenogroupC1viruseshavenotcausedlargeoutbreaksduringthelasttwodecades.ThesubgenogroupC2virusesthatwerereplacedbyB4virusesinTaiwan(China)by2000havecontinuedtobeidentifiedinJapan,SingaporeandThailandoverthelastfewyears.

VietNambegan investigatingEV71 in2005. It is interesting tonote that, althoughC1andC4virusesdocirculateinVietNam,thedominantsubgenogrouphasbeen,andremains,C5(22).ThatsubgenogroupemergedinTaiwan(China)in2006.

AsimilarsituationexistsinChina,wherethesingledominantsubgenogroupC4causedmajoroutbreaksin2008and2009(23,24).ThatsubgenogroupwassampledinChinaandJapaninthelate1990sandearly2000sandwasdominantinTaiwan(China)in2004and2005.It iscurrentlycirculatinginJapan,theRepublicofKorea,Taiwan(China),ThailandandVietNam.TheRepublicofKorearecordedasubgenogroupC3clusterofcasesin2000,whenB4virusesweredominantelsewhereintheregion(25).

Figure2illustratesthetemporaldistributionofthedifferentsubgenogroupsofEV71circulating in various countries in the region. The figure includes data generatedfollowing the enhanced surveillance activities in operation in many countries since1997.Itisthereforeimportanttonotethattherearegapsintheknowledgeregardingcirculatingstrainspriortothatperiod.

Figure 2: Recorded prevalence of EV71 subgenogroups in the Asia-Pacific region

AreaYear

1970s 1980 s 1990s 2000 -2004 2005 -2009

Malaysia

Taiwan

Australia

Japan

Singapore

China

Vietnam

Korea

Mongolia

Thailand

New Zealand

Area-

B1 C2 C4B2

B3

B4

B5

C3

C5

C2B4

C4B5

C4

C1 C4

C2

C1C2 B4

B3 B4C1

B5

B3B4

C4B5C1

C1C2

C4

B5

C5

C5

C1

C1

C2

B1

B2

C2

C2

[ 18 ]


2.2 Virus receptor

Nishimura (26), Yamayoshi (27) and Yang (28), with their colleagues, recentlyindependently demonstrated that human P-selectin glyprotein ligand-1 (PSGL-1),humanscavengerreceptorclassB,member2(SCARB2),andsialic-acid-linkedglycansactasfunctionalreceptorsforEV71byusingdifferenthumancell linesanddifferentcloning strategies. Identification of three structurally and functionally differentreceptors for EV71may provide valuable insights into themolecular basis of EV71infection,includingHFMDandvariousneurologicaldiseases.

2.3 Recombination

Aswithotherenteroviruses,recombinationhasbeenobservedinseveralEV71fieldisolates (2932). It has been observedmost frequently in the 5UTR (untranslatedregion)and3UTRregions;ithasrarelybeenidentifiedinthestructuralproteingeneregion. Although theeffect of recombinationon thevirulenceor transmissibility ofEV71isunknown,laboratorystudieshaveshownthatreplacementofthe3halfofthegenomeofanon-recombinantEV71fieldisolatewithonederivedfromaHEV-Bspeciesvirussignificantly improvesgrowth incellculturecomparedwithanon-recombinantstrain(33).

2.4 Reservoir of EV71

EV71replicatesintheintestinaltractandistypicallyshedforbetweentwoandfourweeks,andsometimesforaslongas12weekspost-infection.Replicationalsooccursintheupperrespiratorytractandthevirushasbeenrecoveredfromthroatswabsforuptotwoweekspost-infection.Thustransmissioncanincludefaecal-oralandrespiratorysecretions through direct person-to-person contact, droplets or fomites. Factorsthataffectthetransmission include levelofhygiene,waterquality,andtheextentofcrowding(34).

Ingeneral,theenteroviruseshaveadistinctseasonalpatternofcirculationthatvariesbygeographicarea.Intropicalandsubtropicalcountries,circulationtendstobeyearround,withmoreoutbreaksintherainyseason.

References

1. McMinn P, et al. Phylogenetic analysis of enterovirus 71 strains isolated during linked

epidemics inMalaysia,Singapore, andWesternAustralia. JournalofVirology,2001,Aug,

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2. CardosaMJ, et al. Molecular epidemiology of human enterovirus 71 strains and recent

outbreaks in the Asia-Pacific region: comparative analysis of the VP1 and VP4 genes.

EmergingInfectiousDiseases,2003,Apr,9(4):461468.

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3. ShimizuH,etal.Molecularepidemiologyofenterovirus71infectionintheWesternPacific

Region.PediatricsInternational,2004,Apr,46(2):231235.

4. Bible JM, et al. Genetic evolution of enterovirus 71: epidemiological and pathological

implications.ReviewsinMedicalVirology,2007,NovDec,17(6):371379.

5. Nix WA, Oberste MS, Pallansch MA. Sensitive, seminested PCR amplification of VP1

sequences for direct identification of all enterovirus serotypes from original clinical

specimens.JournalofClinicalMicrobiology,2006,Aug,44(8):26982704.

6. Oberste MS, et al. Improved molecular identification of enteroviruses by RT-PCR and

ampliconsequencing.JournalofClinicalVirology,2003,Apr,26(3):375377.

7. ObersteMS,etal.Comparisonofclassicandmolecularapproachesfortheidentificationof

untypeableenteroviruses.JournalofClinicalMicrobiology,2000,Mar,38(3):11701174.

8. BrownBA,etal.Molecularepidemiologyandevolutionofenterovirus71strainsisolated

from1970to1998.JournalofVirology,1999,Dec,73(12):99699975.

9. TeeKK,etal.Evolutionarygeneticsofhumanenterovirus71:origin,populationdynamics,

natural selection, and seasonal periodicity of the VP1 gene. Journal of Virology, Apr,

84(7):33393350.

10. HuangSW,et al.Reemergenceofenterovirus71 in2008 in taiwan:dynamicsofgenetic

and antigenic evolution from 1998 to 2008. Journal of Clinical Microbiology, 2009, Nov,

47(11):36533662.

11. IwaiM,etal.GeneticchangesofcoxsackievirusA16andenterovirus71isolatedfromhand,

foot,andmouthdiseasepatientsinToyama,Japanbetween1981and2007.JapaneseJournal

ofInfectiousDiseases,2009,Jul,62(4):254259.

12. MizutaK,etal.Cross-antigenicityamongEV71strainsfromdifferentgenogroupsisolated

inYamagata,Japan,between1990and2007.Vaccine,2009,May21,27(24):31533158.

13. KungSH,etal.Geneticandantigenicanalysesofenterovirus71isolatesinTaiwanduring

1998-2005.ClinicalMicrobiologyandInfection,2007,Aug,13(8):782787.

14. HosoyaM,etal.Geneticdiversityofenterovirus71associatedwithhand,footandmouth

diseaseepidemicsinJapanfrom1983to2003.PediatricInfectiousDiseaseJournal,2006,

Aug,25(8):691694.

15. PodinY,etal.Sentinelsurveillanceforhumanenterovirus71inSarawak,Malaysia:lessons

fromthefirst7years.BMCPublicHealth,2006,6:180.

16. LinKH,etal.EvolutionofEV71genogroupinTaiwanfrom1998to2005:anemergingof

subgenogroupC4ofEV71.JournalofMedicalVirology,2006,Feb,78(2):254262.

17. Sanders SA, et al. Molecular epidemiology of enterovirus 71 over two decades in an

Australianurbancommunity.ArchivesofVirology,2006,May,151(5):10031013.

18. MizutaK,etal.Frequentimportationofenterovirus71fromsurroundingcountriesintothe

localcommunityofYamagata,Japan,between1998and2003.JournalofClinicalMicrobiology,

2005,Dec,43(12):61716175.

[ 20 ]


19. FujimotoT,etal.Outbreakofcentralnervoussystemdiseaseassociatedwithhand,foot,and

mouthdiseaseinJapanduringthesummerof2000:detectionandmolecularepidemiologyof

enterovirus71.MicrobiologyandImmunology,2002,46(9):621627.

20. ChuPY,etal.Molecularepidemiologyofenterovirus71 inTaiwan.ArchivesofVirology,

2001,146(3):589600.

21. ShimizuH,etal.Enterovirus71fromfatalandnonfatalcasesofhand,footandmouthdisease

epidemics in Malaysia, Japan and Taiwan in 1997-1998. Japanese Journal of Infectious

Disease,1999,Feb,52(1):125.

22. TuPV, et al.Epidemiologic and virologic investigation of hand, foot, andmouth disease,

southernVietnam,2005.EmergingInfectiousDiseases,2007,Nov,13(11):17331741.

23. YangF,etal.Enterovirus71outbreakinthePeoplesRepublicofChinain2008.Journalof

ClinicalMicrobiology,2009,Jul,47(7):23512352.

24. ZhangY,etal.Anoutbreakofhand,foot,andmouthdiseaseassociatedwithsubgenotype

C4 of human enterovirus 71 in Shandong,China. Journal ofClinicalVirology, 2009,Apr,

44(4):262267.

25. JeeYM,etal.GeneticanalysisoftheVP1regionofhumanenterovirus71strainsisolated

inKoreaduring2000.ArchivesofVirology,2003,Sep,148(9):17351746.

26. Nishimura Y, et al. Human P-selectin glycoprotein ligand-1 is a functional receptor for

enterovirus71.NatureMedicine,2009,Jul,15(7):794797.

27. YamayoshiS,etal.ScavengerreceptorB2isacellularreceptorforenterovirus71.Nature

Medicine,2009,Jul,15(7):798801.

28. YangB,ChuangH,YangKD.Sialylatedglycansasreceptorandinhibitorofenterovirus71

infectiontoDLD-1intestinalcells.VirologyJournal,2009,6:141.

29. ChenX, et al.Analysis of recombination andnatural selection inhumanenterovirus71.

Virology,2010,Mar15,398(2):251261.

30. DingNZ,etal.Appearanceofmosaicenterovirus71inthe2008outbreakofChina.Virus

Research,2009,Oct,145(1):157161.

31. HuangSC,etal.Appearanceofintratypicrecombinationofenterovirus71inTaiwanfrom

2002to2005.VirusResearch,2008,Feb,131(2):250259.

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33. PhuektesP.DevelopmentofareversegeneticssystemforHumanenterovirus71(HEV71)

and the molecular basis of its growth phenotype and adaptation to mice. PhD Thesis,

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Diseases,2002,Sep,8(9):995997.

[ 21 ]



Section 3: Laboratory Diagnosis

3.1 Overview

Rapid identification of the causative agent of HFMD, particularly whether there isEV71 infection, can help clinicalmanagement by focusing attention on the possiblecomplications.WhenpatientspresentwithothermanifestationsofEV71,suchasCNSdiseaseorcardiovascularcollapse,rapidvirologicaldiagnosisisevenmorehelpfulbecauseofthebroaddifferentialforthoseconditionsandthespecifictreatmentsavailable(1).IdentificationoftheagentsresponsibleforoutbreaksofHFMDisalsoimportantwithregardstopredictingtheseverityoftheoutbreakandinitiatingappropriatepublichealthinterventions. Aswithotherenteroviruses, confirmatorydiagnosishas traditionallybeenbasedoncellcultureandvirusisolationandidentification,bothofwhicharetime-consumingandresource-intensive.Modernmoleculartechniqueshavebeendevelopedandarenowinuseinsomelaboratories.Effectiveandaccuratevirologicaldiagnosisdependsonthecorrecttimingandcollectionofappropriateclinicalspecimens,andtheirtransporttothelaboratoryunderoptimalconditions.Thisrequiresclosecooperationbetweenlaboratoryexperts,epidemiologistsandclinicians.

3.2 Laboratory safety

Toensuresafehandlingofclinicalsamplesandinfectiousmaterials,laboratoriesshouldfollowthenecessarybiosafety,chemical,fireandelectricalsafetyrequirementstoprotectstaff,thecommunityandtheenvironment(2).Thedirectorsandallstaffofdiagnosticlaboratories should be familiar with international and local biosafety requirements,includingsafehandlingandtransportationofclinicalsamplesandinfectiousmaterials,including goodmicrobiological techniques and basic biosafety level of the facilities,normallyBSL-2forprimaryisolation(3).

3.3 Clinical samples

Throatandvesicle(ifavailable)swabsamplesinvirustransportationmedium(VTM)areconsideredtobethemostusefulspecimensintermsoftherateofvirusdetectionofHFMD(4)andintermsofbothinpatientandoutpatientavailability. EV71canbeshedinthestoolforseveralweeksandstool(rectalswab)samplesarealsoappropriateclinicalspecimensforvirusdetectionand/orisolation.

[ 22 ]


The risk of recent coincidental infection, rather than casual infection, should beconsidered forsomeclinicalsamples,particularly forstoolsamples. Vesicle isolatesare the most useful, because they always represent current systemic infection (4).Given thepotentialnumberofsamples, from largenumbersofpatients, laboratoriescanbecomeoverwhelmed.

To increase the possibility of enterovirus detection, collecting throat swab samplesforallpatientsplusswabsfromat leasttwovesiclesor fromtherectumforpatientswithnovesiclesisrecommended.ThevirusdetectionratesofEV71andCA16fromcerebrospinalfluid(CSF)samplesareratherlow(lessthan5%)(4,5).However,CSFsamples are useful for virus detection of other enteroviruses, particularly forHEV-B-associated aseptic meningitis. Serum samples from inpatients may be useful forserologicaldiagnosis,butthereliabilityofserologicaltestsforEV71infectionrequirescarefulevaluation.

3.4 Laboratory diagnosis methods

Asforotherenterovirusinfections,includingpoliomyelitis,confirmeddiagnosesbasedoncellculture,virusisolationandidentificationofenterovirusesisstillthestandardmethodforlaboratorydiagnosis.Whilevirusisolatesareneededforfurthermolecularcharacterizations of EV71, virus isolation and identification are generally laboriousand time-consumingandsuchmethodsarenotpractical forclinicaldecision-making.Although rapid laboratorydiagnosticmethodsusing clinical specimens are available,standardizationofthemethodsandcomparativeevaluationofthereliabilityoftherapidtestsarestillneeded.

Virus isolation

Someclinicalspecimensrequireappropriatepre-treatment(completemixing,filtration,chloroformtreatment)beforeinoculation.Anumberofhumanandnon-humanprimatecelllinesareavailableforvirusisolationandidentificationofenteroviruses.Ofthesecelllines,RD(humanrhabdomyosarcomacells)andVero(Africangreenmonkeykidneycells)cellshavebeenwidelyusedforvirusisolationofEV71andCA16becauseoftheirrelativelyhighsensitivityandtheapparentcytopathiceffect(CPE) inducedbyEV71andCA16.RDcellsareavailable fromtheGlobalPolioLaboratoryNetworkand thequalitycontrolof thecells is routinelycarriedoutaccording to thePolioLaboratoryManual(3)toensuresensitivityagainstpolioviruses.Thequalityofcellculturesusedinvirologicalinvestigationisimportantforthestandardizationofenterovirusisolationanditscharacterization.

It is important to use several different human and non-human primate cell lines toincreasethepossibilityofvirusisolationincellcultures.Additionalcelllines(includingMRC-5,HEL,HeLa, L20B) can also be used for virus isolation of EV71 and other

[ 23 ]



enteroviruses, including polioviruses. Although the receptor-specificities of EV71isolatesandtheotherHEV-Astrainswouldneedtobecharacterized,mousecelllinesexpressing functional cellular receptors for EV71 (human P-selectin glycoproteinligand-1 andhuman scavenger receptor classB,member 2)would beuseful for theselectiveisolationofEV71and/orCA16fromclinicalspecimens(6,7).Sucklingmice(less than 48 hours old) are still useful for virus isolation of someHEV-A isolates,particularly forclinicalsamples fromherpanginacases (8). However,virus isolationandidentificationusingsucklingmiceistime-consumingandrequiresspecifichumanandequipmentresources.

Identification of virus isolates

Neutralization test

Following virus isolation in cultured cells, the serotype of EV71 and CA16 isolatescan be identified by a conventional neutralization test with a qualified type-specificantiserum. The type-specific antisera against HEV-A strains, including EV71 andCA16,arenotcommerciallyavailableandthesupplyofin-houseantiseraisthereforegenerally limited. The neutralization test is still a reliablemethod for enterovirusidentification,butitcantakeuptoaweek(57days)tocompletetheassay.

Reverse transcription polymerase chain reaction (RT-PCR) and sequencing

Following virus isolation in cultured cells, reverse transcription polymerase chainreaction(RT-PCR)amplificationofviralRNAandsequencingofthedeoxyribonucleicacid(DNA)amplicons,usingvariousgenetargets(5untranslatedregion[5UTR],VP1,VP4/VP2 genes), have been widely used for molecular identification of enterovirusisolates. The advantage of the RT-PCR and sequencing strategy is the universaldetection and amplification of enterovirus gene targets, regardless of the serotypesandgenotypesofenteroviruses,possibly includingnewlyemergedvariantsandnewserotypesof enteroviruses (911). While sequencingof the shorterVP4 region (orVP4pluspartialVP2)issufficientforregularsurveillance,confirmationofthedataformolecular epidemiological research should use theVP1 region of the genome.Thisis also important as theVP1gene is themostexposedand immunodominantof thecapsid proteins and is thereforemost likely to change in response to immunogenicpressure(12,13).TheRT-PCRandsequencingstrategymaydependonroutineaccessto the sequencing facility at diagnostic laboratories and requires higher installationandrunningcosts. Specific trainingactivities for laboratoryexpertsand techniciansonmolecularidentificationofenterovirusesareneededtominimizetheriskofcross-contaminationandtoensurethereliabilityofidentification,includingsequenceanalysisanddatamanagement.

EV71-specific RT-PCR

Following virus isolation in cultured cells,RT-PCR amplification of viralRNAusingEV71-specific primers is useful for themolecular identification of EV71 (14). The

[ 24 ]


EV71-specific RT-PCR amplification is a rapid and simple identificationmethod andrequires lower installation and running costs. EV71-specific primers should berevisedaccordingtothesequencesofnewlyemergingEV71genogroupsandvariantsto maintain the reliability of the EV71-specific RT-PCR test (14). General trainingactivities for laboratory technicians on RT-PCR are needed tominimize the risk ofcross-contamination.

Indirect immunofluorescence assay

Indirectimmunofluorescenceassay(IFA)testsusinganti-EV71monoclonalantibodiescan provide rapid, presumptive EV71 identification. The IFAmethod is technicallysimpleandrapid,andtheantibodiesarecommerciallyavailable(15),althoughrelativelyexpensivetopurchase.

Rapid diagnosis directly from clinical samples

Nested RT-PCR

Targeting of conserved 5UTR has been widely used for the universal detection ofenteroviruses. However, 5UTR RT-PCR methods are inappropriate for serotype-specific detection and identification of EV71. Technical improvement of RT-PCRmethodsbasedontheVP1regionhasenabledpartialVP1sequencingusingconsensus-degenerate hybrid oligonucleotide primer (CODEHOP) with a high sensitivity andbroader specificity for all known enterovirus serotypes (16). Enteroviruses canthereforebeidentifiedbyVP1sequencesderivedfromtheCODEHOPPCRproductsdirectlyfromclinicalsamples.MultiplexRT-PCRmethodshavebeendevelopedfortheidentificationofEV71andCA16(17)andforthespecificdetectionofEV71andCA16directlyfromclinicalsamples(18).

Real-time PCR

Real-timeRT-PCRsystemshaverecentlybecomeavailabletodiagnosticlaboratoriesforrapidandspecificdetectionofanumberofviralinfectiousdiseases.One-tubereal-time RT-PCR systems can reduce the risk of cross-contamination when comparedwithconventionalRT-PCR,particularlynestedPCRsystems.WhileseveraldifferentEV71-specificreal-timeRT-PCRsystemshavebeenreported(1921)thereliabilityofsuchsystemsneedstobeaddressedusingdifferentgenogroupsofEV71,CA16,HEV-Astrains, and clinical samples. In general, EV71-specific real-time RT-PCR primersand probes should be revised according to the sequences of newly identified EV71genogroupsandvariantstomaintainthereliabilityofEV71-specificreal-timeRT-PCR.

New diagnostic approaches

InadditiontoEV71-specificreal-timeRT-PCRsystems,severalotherpromisingoptionsforrapidandconvenient(bedside)diagnosisareunderinvestigation. Althoughrapidviralantigen-detectionkitsusing immunochromatographyarecommerciallyavailablefor a number of viral infectious diseases, EV71 antigen-detection kits have not yet

[ 25 ]



beendeveloped,mainlyduetothelowsensitivityfromclinicalsamples(22).Whilethereversetranscriptionloop-mediatedisothermalamplification(RT-LAMP)methodcanbeusedasarapidandsensitiveisothermaldetectionmethodforenteroviruses,itisnotspecifictocertainserotypes(genotypes)ofenterovirus(23).

Evaluation of the reliability of new laboratory diagnostic methods

To encourage the development of rapid and convenient (bedside) diagnostic kits forEV71infectionintheregion,astandardizedevaluationsystemtocomparethesensitivityand specificity of diagnostic methods is critical. For the evaluation of molecularidentificationmethods,establishmentofanEV71strainbank,containingallavailableEV71subgenogroups(A,B1-B5,C1-C5,plusnewlyemerginggenogroups),shouldbeconsidered.Linkingthistoaqualityassurancesystemandnetworkingoflaboratorieswouldensurestandardsforidentificationofenterovirusesandtheirsubgroupscanbeappliedinthefuture.

Molecular epidemiological analysis (genotyping) of EV71 strains

A powerful tool for tracking the circulation of EV71 strains is the molecularcharacterizationofEV71genomes(genotyping).BycomparingtheextentofgeneticchangesthatareobservedbetweenEV71strains,thegeographicandevolutionaloriginofaviruscanbedetermined. BaseduponacapsidVP1sequencedatabaseofEV71strains,itispossibletodeveloprapidapproachestotrackingtheregionaltransmissionand current prevalence ofEV71 strains (see the section onVirology). For detailedmolecularepidemiological analysisofEV71strains, theentireVP1sequenceshouldbedeterminedandanalysed, althoughpartialVP1sequencingwouldbesufficient togenotypeeachEV71isolate.

Serological analysis

Testingforneutralizingantibodiesagainstenterovirusesisnotrecommendedforroutineuseinthediagnosisofenterovirusinfections.Pairedserumsamplesarecollectedforserologicaldiagnosisofcertainserotypesofenterovirus,but interpretationofserumantibodytitersissometimesdifficult.Ontheotherhand,theneutralizingtestagainstEV71 would be useful for evaluating of immunity levels for EV71 infection withincommunities (23,24),aswellas formonitoringthecross-reactivityofserumamongdifferentgenogroupsofEV71(25).

SerumsamplesfrominpatientscouldbeusefulfortherapidimmunoglobulinM(IgM)detectionofEV71 (22,26),but thespecificityandsensitivityofserological tests forEV71infectionremainsanareawherecarefulevaluationisneeded.

[ 26 ]


References

1. Ooi MH, et al. Clinical features, diagnosis and management of human enterovirus 71

infection.LancetNeurology,2010,9(11):10971105

2. Laboratorybiosafetymanual,3rdedition.Geneva,WorldHealthOrganization,2004.

3. Poliolaboratorymanual,4thedition.Geneva,WorldHealthOrganization,2004.

4. OoiMH,etal.Evaluationofdifferentclinicalsampletypesindiagnosisofhumanenterovirus

71-associated hand-foot-and-mouth disease. Journal of Clinical Microbiology, 2007, Jun,

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5. ChanKP,etal.Epidemichand, footandmouthdiseasecausedbyhumanenterovirus71,

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Medicine,2009,Jul,15(7):798801.

8. ShimaT,etal.Enterovirusdetectionstatusfrompatientswithherpanginaandhand,foot

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9. ObersteMS,etal.TypingofhumanenterovirusesbypartialsequencingofVP1.Journalof

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10. ObersteMS,etal.Molecularevolutionofthehumanenteroviruses:correlationofserotype

withVP1sequenceandapplicationtopicornavirusclassification.JournalofVirology,1999,

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11. BrownBA,etal.Molecularepidemiologyandevolutionofenterovirus71strainsisolated

from1970to1998.JournalofVirology,1999,Dec,73(12):99699975.

12. CardosaMJ, et al. Molecular epidemiology of human enterovirus 71 strains and recent

outbreaks in the Asia-Pacific region: comparative analysis of the VP1 and VP4 genes.

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humanenteroviruses.JournalofMedicalVirology,2010,Apr,82(4):649657.

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humanenterovirus71:improvedprimersforthespecificdetectionofhumanenterovirus71

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16. Nix WA, Oberste MS, Pallansch MA. Sensitive, seminested PCR amplification of VP1

sequences for direct identification of all enterovirus serotypes from original clinical

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17. Chiueh TS, et al. Multiplex Reverse Transcription-semi-nested PCR for Differentiating

Enterovirus71,CoxsackievirusA16,andpoliovirusesfromotherenteroviruses.Journalof

MedicalSciences,2001,21(6):277282.

18. ChenTC,etal.Combiningmultiplexreversetranscription-PCRandadiagnosticmicroarray

to detect and differentiate enterovirus 71 and coxsackievirus A16. Journal of Clinical

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real-time RT-PCR hybridization probe assay. Molecular and Cellular Probes, 2006, Apr,

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20. TanEL,etal.Rapiddetectionofenterovirus71byreal-timeTaqManRT-PCR.Journalof

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21. XiaoXL,et al.Simultaneousdetectionofhumanenterovirus71 andcoxsackievirusA16

in clinical specimens bymultiplex real-time PCRwith an internal amplification control.

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22. Wu H-S (Centers for Disease Control, Taiwan[China]). Personal communication to H.

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23. AritaM,etal.Developmentofareversetranscription-loop-mediatedisothermalamplification

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26. Wang SY, et al. Early and rapid detection of enterovirus 71 infection by an IgM-capture

ELISA.JournalofVirologicalMethods,2004,Jul,119(1):3743.

[ 28 ]


Section 4: Pathogenesis in EV71 Infection

4.1 Overview

There is very little information concerning the pathology of uncomplicated EV71-associatedHFMDandherpangina. Inamousemodel,theviruswasshowntoinfectskin, suggesting that human skin or oralmucosa lesions could also be the result ofdirectinfectionofsquamouscells(1).Whileitispossiblethatpathologicalchangesareconfinedtoskinrashesandoral lesions,there isa lackof informationavailable fromskinororalmucosabiopsies.

The neurovirulence factors of EV71 in human infections are still unknown. So far,thereisnoconvincingevidenceofspecificvirusgenomemutationsthatmayfacilitateinfectionofthehumancentralnervoussystem(CNS).Otherfactors,suchashigherinitialinfectivedosesleadingtoseveredisease,arealsopossible.Certaincytokines,chemokines, alterations of cell mediated immunity and human leukocyte antigen(HLA) subtypes appear tobe associatedwith ahigher incidenceof cardiopulmonarycomplications.Therelationshipbetweenpathogenesisanddistributionofviralentryreceptors(scavengerreceptorB2,P-selectinglycoproteinligand-1andsialicacid-linkedglycans)andhostfactors,suchasgenderandagegroup,isunknown.

ThepathologicalfindingsintheCNSinfatalcasesofEV71arestereotypedandconsistof neuronophagia, perivascular cuffing, focal oedema and infiltration of inflammatorycells.Viralcytolysisappearstobeanimportantmechanismforneuronaldamage.Themost severe inflammation is found in thehypothalamus,brain stem, spinal cordandcerebellardentatenucleus.Medullaryinflammationandfindingsinthelungsandheartareconsistentwithneurogenicpulmonaryoedemaasacauseofdeath.AnimalstudiesconfirmsomeofthehumanautopsyfindingsandsuggestthatmotorpathwaysmayplayanimportantroleinviraltransmissionintheCNS.

4.2 Virus neuro-virulence factors

Viral genome mutations

To date, no convincing evidence of specific virus genome mutations that conferneurovirulenceinhumanshasbeenreported.

[ 29 ]



However, clinical epidemiological evidence from outbreaks in Perth, Australia, andSarawak, Malaysia, suggests that there may be differences between genogroups intermsofbiologicalbehaviourandvirulence(2).InPerth,in1999,subgroupsB3andC2werebothcirculating(3,4).

C2 viruses were linked to the 1998 epidemic in Taiwan (China) and were almostexclusively isolated fromchildrenwithsevereneurologicaldisease;onlyone isolatecamefromacaseofuncomplicatedHFMD(3,4).Bycontrast,B3virusesthatweresimilartothosefromthe1997Sarawakepidemic,wereisolatedmainlyfromchildrenwithuncomplicatedHFMDorasepticmeningitis,orthosewithneurologicalcomplications,noneofwhomdied(5).IntwodiscreteepidemicsinSarawakinwhicheitherB4orB5viruseswerepredominant, a studyof 277 childrenwithEV71-associatedHFMDshowedthatB4viruseswerelesslikelythanB5virusestocauseCNSinfectionorbepartofafamilycluster(6).

More extensive virological surveillance and genetic analysis from the region mayprovide further evidence of potential genetic determinants of virulence. Animalstudies, however, have demonstrated that there are four co-expressed mutationsderivedfromtheSabinpoliovirustype1attenuatedEV71inbothcynomolgusmonkeys(7)andinnon-obesediabetic/severecombinedimmunodeficient(NOD/SCID)mice(8).A3Dpolymerasegenemutationhasbeenshowntointroduceatemperature-sensitivephenotypeincellcultureandanattenuationofvirulenceinnewbornmice(9).

Other factors

Otherfactors,suchashigherinfectivevirusdosesleadingtomoresevereillness,arepossibleandshouldbeinvestigatedfurther.

4.3 Host factors

Immunopathology

There have been relatively few studies of the human immune response to EV71infection. Induction of specific cytokines and chemokines has been associated withcertainadverseoutcomesofEV71infection,mostnotablypulmonaryoedema.Abnormalelevationofcytokinesmaybeonemechanismforpulmonaryoedemainfatalcases.Inatleasttwostudies,cerebrospinalfluidlevelsofinterleukin(IL)-6werefoundtoberaisedin caseswith pulmonary oedema (10, 11). Higher levels of interferongammawerealsofoundincerebrospinalfluidinsimilarcases(11).LevelsofIL-6,tumournecrosisfactoralpha,IL-1beta(12)andIL-10(11)werealsofoundtobehigherintheserumofpulmonaryoedemacases.Furthermore,levelsofchemokines,IL-8,interferoninducedprotein(IP-10),monocytechemoattractantprotein(MCP-1)andmonokineinducedbyinterferongamma(MIG)werefoundtobehigherinserum,andMIGinbothserumandcerebrospinalfluidinpulmonaryoedema(13).

[ 30 ]


Withregardstocell-mediatedimmunity,areductionofTlymphocytesandNKcellshasbeenreportedinpatientswithpulmonaryoedema(14).Anotherstudydemonstratedreducedproductionofinterferongamma,IL-1beta,IL-6andmacrophageinflammatoryprotein-1alphainstimulatedperipheralmononuclearcellsinpatientswithpulmonaryoedema(15).

HLA-A33isreportedtobeassociatedwithsusceptibilitytoEV71infection,particularlyinAsianpopulationswheretheHLAprevalence(17%-35%)ishigherthaninCaucasianpopulations. In addition, HLA-A2 may possibly be linked to increased risk ofcardiopulmonarycomplications(16).

Virus receptors on host target cells

In 2009, three receptors were identified that facilitate virus entry into host cells:scavengerreceptorB2(17)andP-selectinglycoproteinligand-1(18).Respectively,thosereceptorshavebeenreported tobeubiquitouslyexpressedor limited to leukocytes.Whether thepresenceorabsenceof thosereceptors inhuman tissuesmight impactonviralpathogenesishasnotbeeninvestigated. Similarly,viralreplicationsitesandviraltransmissionrouteswithinthebodymayberelatedtothepresenceorabsenceofreceptorsinsusceptibletissues.

Other host factors

Otherhost factorsthatmaybeimportant forsusceptibilityto infectioncould includegender, as a higher incidence of severe diseasehas been reported inmale children.Moreover,therelativeimmaturityoftheinnateandadaptiveimmunesysteminchildrenagedbetweenoneandfiveyearscouldaccountforthehigherincidenceofneurologicalcomplicationsinthatagegroup.Evidenceforthisisstilllacking.

4.4 Pathological findings

Human autopsies

Autopsies conducted in mainland China, Malaysia, Singapore and Taiwan (China)have been useful in improving understanding of the pathogenesis of severe diseaseand theunderlyingpathological insult leading todeath. The inflammatory responseseeninEV71encephalomyelitisistypicalofviralencephalitides,thefeaturesofwhichinclude neuronophagia, perivascular cuffing, focal oedema andmacrophage/microgliainfiltration(Figure3)(19-22).Themainareasofinflammationappeartobelocalizedtothehypothalamus,brainstem,spinalcordandcerebellardentatenucleus.Althoughmildinflammationcanbeseeninthecerebralcortex(especiallythemotorcortex),itis entirely absent from the anterior pontine nuclei and cerebellar hemisphere. Thetopographicdistributionofinflammationandvirussuggestsretrogradeperipheralmotornerveviral spread into theCNS. Within theCNS,otherneuralpathways, includingmotorpathways,couldbeinvolved(19).Viralantigens/RNAhavebeendemonstrated

[ 31 ]



in neuronal body and processes (Figure 4), and inmacrophages participating in theneuronophagiaseenininflamedareas.Thisprovidesevidencethatneuronsarethemainviraltargetsandthatviralcytolysisisanimportantmechanismforneuronalinjury.Themechanismcouldinvolveapoptosis,asthevirusisabletoinduceapoptosisinneuronalandglialcellcultures (23,24). Pathologicalevidenceofmedullary inflammationanddestructionisconsistentwithneurogenicpulmonaryoedemaastheprimarycauseofdeath.

Lung findings at autopsywere found to be consistent with interstitial pneumonitis,pulmonaryoedemaandhaemorrhage,andmyocardialcongestion(20).Otherstudieshavefoundthat,apart frompulmonaryoedema,therewasnoevidenceofpulmonaryinflammationorviralantigeninthelungparenchyma(25).Myocarditisandmyocardialinfectionaregenerallyabsent. Thepathogenesisofdeathresulting frompulmonaryoedemaaloneorpulmonaryoedemawithconcurrentpulmonaryhaemorrhageduringEV71infectionisnotcompletelyunderstood.Basedonthefindingsabove,however,myocarditisisunlikelytobethecauseofdeath.

Animal models

Monkey(26,27)andmousemodels(1,28-30)havebeenusedtostudytheneurovirulenceof EV71. In such models, the CNS pathology has, so far, confirmed that EV71 isneuronotropic.Amajordrawbackofthemonkeymodelsisthatmonkeysarerefractorytoinfectionbytheoralroute.

Figure 3:

Perivascularcuffingandparenchymal

infiltrationbyinflammatorycellsin

thehumanmedullainEnterovirus71

encephalomyelitis(Haematoxylinandeosin

stain(H&E),magnificationx10objective)

Figure 4:

Enterovirus71-infectedneuronshowing

cytoplasmicviralRNA(insituhybridization,

magnificationx40objective)

[ 32 ]


InonemousemodelofEV71encephalomyelitis, thedistributionofvirus infiltrationandinflammationwassimilartothatseeninhumanencephalomyelitis,suggestingthatmotor pathways are important for viral transmission into and within the CNS (30).Viralantigenswerefoundintheskeletalmuscleandbrownfat,butthishasnotbeenreportedinhumanEV71infections.InfectivitystudiesshowthattheskeletalmusclesaremajorprimaryvirusreplicationsitesforEV71inmiceandthatmyositisisaprimarycauseofdeath(29,30).Thecurrentlyavailableanimalmodelscouldbeusedforfurtherinvestigationofdiseasepathogenesisandforanti-viraldrugandvaccinetesting.

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[ 35 ]



Section 5: Clinical F

Documents

Guidance for the Clinical Management of h Fmd